PERSPECTIVE ARTICLE
BIOENGINEERING AND BIOTECHNOLOGY
published: 28 August 2014
doi: 10.3389/fbioe.2014.00030
Cofactor engineering for enhancing the flux of metabolic
pathways
M. Kalim Akhtar 1 * and Patrik R. Jones 2 *
1
2
Department of Biochemical Engineering, University College London, London, UK
Department of Life Sciences, Imperial College London, London, UK
Edited by:
Pablo Carbonell, University of Evry,
France
Reviewed by:
Juan Manuel Pedraza, Universidad de
los Andes, Colombia
Gary Sawers, Martin-Luther
University Halle-Wittenberg, Germany
*Correspondence:
M. Kalim Akhtar , Department of
Biochemical Engineering, University
College London, Torrington Place,
London, WC1E 7JE, UK
e-mail: [email protected] ;
Patrik R. Jones, Department of Life
Sciences, Imperial College London,
Sir Alexander Fleming building,
London, SW7 2AZ, UK
e-mail: [email protected]
The manufacture of a diverse array of chemicals is now possible with biologically engineered
strains, an approach that is greatly facilitated by the emergence of synthetic biology. This
is principally achieved through pathway engineering in which enzyme activities are coordinated within a genetically amenable host to generate the product of interest. A great deal
of attention is typically given to the quantitative levels of the enzymes with little regard to
their overall qualitative states. This highly constrained approach fails to consider other factors that may be necessary for enzyme functionality. In particular, enzymes with physically
bound cofactors, otherwise known as holoenzymes, require careful evaluation. Herein, we
discuss the importance of cofactors for biocatalytic processes and show with empirical
examples why the synthesis and integration of cofactors for the formation of holoenzymes
warrant a great deal of attention within the context of pathway engineering.
Keywords: cofactors, metabolic pathway engineering, Fe–S clusters, enzymatic activity, synthetic biology
INTRODUCTION
Synthetic biology permits the engineering of biological devices or
systems with novel or enhanced functions (Church et al., 2014).
Such an approach has numerous applications, most notably in the
manufacture of a diverse number of molecules including household chemicals, biofuels, and pharmaceutical drugs (Keasling,
2010). This is principally achieved through pathway engineering
in which enzyme activities are carefully coordinated to generate
the product of interest. Approaches based on the activities of isolated enzymes (in vitro) or whole cells (in vivo) can be employed for
this purpose (Guterl et al., 2012; Stephanopoulos, 2012). The latter
approach in particular offers a significant benefit with respect to
complex, multi-step pathways that rely on secondary cellular factors, as well as additional pathway processing. Since the enzymes
serve as the workhorse components of these biocatalytic systems,
both their quantitative levels and qualitative states are important
parameters to consider for pathway engineering. A great deal of
focus is typically placed on the quantitative levels of the enzyme
(Zelcbuch et al., 2013) with little attention given to their overall
qualitative states. Thus, the assumption is usually made that these
enzyme components are functioning at full capacity. However, this
may not always be the case given that a large subset of enzymes
depend on cofactors for functionality.
SIGNIFICANCE OF COFACTORS IN BIOLOGY
All biological organisms possess a network of pathways that lead to
the production of metabolites with an array of cellular functions
(Feist et al., 2009). The synthesis and interconversion of these
metabolites is made possible by the catalytic activities of countless enzymes. By lowering the activation energy barrier, enzymes
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catalyze reactions at considerably faster rates than their chemical
counterparts. This characteristic property along with the relatively
high degree of substrate selectivity, permit the use of enzymes
as catalysts for industrial purposes. For those enzymes, which
rely solely on amino acids for catalysis, the types of reactions
are extremely narrow in scope and, for the most part, restricted
to acid/base and electrophilic/nucleophilic reactions (Broderick,
2001). To further extend, the range of reactions, enzymes are commonly associated with non-protein moieties known as cofactors
(Broderick, 2001).
In the broadest sense of the term, cofactors are thought to be
associated with well over half of known proteins (Fischer et al.,
2010a). However, for this article, the term “cofactor” will refer
specifically to those moieties, either organic or inorganic, which
remain physically associated with the enzyme throughout the
catalytic cycle (Fischer et al., 2010a). This excludes dissociable
cosubstrates such as NADPH and glutathione. Additionally, since
the primary focus of this article is on pathway engineering, only
those cofactors that require de novo pathways for syntheses will be
mentioned and sole metal entities such as calcium and selenium
will also be excluded. Using this strict definition, a selection of
common cofactors are listed in Table 1. These are categorized into
two types: organic and inorganic (Rees, 2002; Fischer et al., 2010b).
Members of the organic group of cofactors tend to be derivatives
of vitamins and undertake numerous types of reactions, while
the inorganic group is usually based on various arrangements of
iron–sulfur (Fe–S) clusters.
In its cofactor-bound state, enzymes are referred to as holoenzymes while in the unbound state, they are known as apoenzymes
(Figure 1A). Two discrete structural parts are required for the
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Cofactor engineering for metabolic pathways
Table 1 | Examples of enzyme bound cofactors.
Enzyme bound cofactor
Type of reaction catalyzed
Example of a cofactor-containing enzyme
Associated pathway
Biotin
Carbon dioxide addition
Acetyl CoA carboxylase
Fatty acid biosynthesis
Factor F430
Methyl transfer
Methyl coenzyme M reductase
Methanogenesis
Flavin mononucleotide
Electron transfer
Cytochrome P450 reductase
Detoxification
Heme
Electron transfer
Cytochrome P450
Detoxification
Lipoic acid
Acyl/methyl amine transfer
2-oxoacid dehydrogenase
Citric acid cycle
MIO cofactor
Carbon–hydrogen bond activation
Phenylalanine ammonia-lyase
Polyphenol biosynthesis
Molybdopterin
Electron transfer
Xanthine oxidase
Purine catabolism
Phosphopantetheine
Acyl carrier
Carboxylic acid reductase
Fatty acid metabolism
Pyridoxal 50 phosphate
Transamination
Glycogen phosphorylase
Glycogenosis
Pyrroloquinoline quinone
Electron transfer
Methanol dehydrogenase
Methane metabolism
Thiamine pyrophosphate
Carbon dioxide removal
Pyruvate ferredoxin/flavodoxin reductase
Pyruvate decarboxylation
Topaquinone
Amine oxidation
Amine oxidase
Urea cycle
Fe–S
Electron transfer
Ferredoxin
Iron–sulfur cluster biogenesis
H-cluster
Hydrogen activation
Fe–Fe hydrogenase
Hydrogen metabolism
Fe-Moco
Nitrogen reduction
Nitrogenase
Nitrogen fixation
C-cluster
Carbon monoxide oxidation
Carbon monoxide dehydrogenase
Carbon monoxide metabolism
P-cluster
Electron transfer
Nitrogenase
Nitrogen fixation
ORGANIC COFACTORS
INORGANIC COFACTORS
For a more comprehensive list of organic cofactors refer Fischer et al. (2010b).
synthesis of a holoenzyme: a polypeptide chain and a cofactor
moiety. The former is generated by the ubiquitous translational
machinery while the latter, depending upon the cofactor, is synthesized by a defined metabolic pathway. In certain cases, subtle
variations of the cofactor pathway may exist. For example, in animals, fungi, and α-proteobacteria, heme synthesis is initiated by
5-aminolevulinate synthase, via condensation of glycine and succinyl CoA, while in photosynthetic eukaryotes and some species
of the α-proteobacterial group, it depends on glutamate, via the
concerted actions of three enzymes (Layer et al., 2010). Once synthesized, the cofactor is integrated with the apoenzyme, either
in a co-translational or post-translational manner, to form the
holoenzyme. The nature of the association may be covalent and,
in such cases, linkages are typically formed with serine, threonine,
histidine, tyrosine, and lysine residues and catalyzed by a distinct
maturation system. As an example, the heme c in cytochrome c
is attached to a cysteine residue, via the vinyl group with the aid
of Ccm (cytochrome c maturation) factors (Sanders et al., 2010).
Alternatively, the interaction may be tight but non-covalent as in
the case of the flavin-containing enzyme, acyl CoA dehydrogenase
(Thorpe and Kim, 1995).
IMPORTANCE OF COFACTOR SYNTHESIS FOR BOTH ENZYME
AND PATHWAY FUNCTIONALITY
As integral components of numerous holoenzymes, cofactors are
required for the majority of metabolic pathways. To name but a
few, these include: FAD in succinate dehydrogenase for the Krebs
cycle; TPP in transketolase for the pentose phosphate pathway;
pyridoxal phosphate in glycogen phosphorylase for glycogenolysis;
H-clusters in hydrogenases for hydrogen metabolism; Fe–MoCo in
nitrogenases for nitrogen fixation; biotin in acetyl CoA carboxylase
for fatty acid biosynthesis; and haem in cytochrome P450 for
Frontiers in Bioengineering and Biotechnology | Synthetic Biology
FIGURE 1 | (A) Generalized overview of the synthesis of holoenzymes.
(B) Significance of cofactor engineering for enhancing the output of
holoenzyme-dependent pathways. The example (Akhtar and Jones, 2009)
illustrates a synthetic pyruvate:H2-pathway that is heavily dependent on
Fe–S clusters. These clusters are required for (i) the proteins/enzymes
directly involved in the pathway for hydrogen production and (ii) the
maturation factors that are responsible for the synthesis and integration of
the H-cluster present in Fe–Fe hydrogenases.
various detoxification pathways. The most crucial point to consider is that the functional output of holoenzymes can only arise
if the apoenzyme is correctly folded with its cofactor. Without the
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Akhtar and Jones
cofactor, such enzymes would be rendered inoperable and the associated pathways would become redundant. This situation, though
undesirable, is likely to be encountered in a bottom-up approach
to microbial engineering in which a genetically amenable and wellcharacterized organism, such as Escherichia coli and Saccharomyces
cerevisiae may be completely devoid of cofactors necessary for heterologous enzyme activity. Alternatively, the capability of the host
to synthesize the required cofactor exists but may be insufficient,
resulting in a pool of enzymes with a high ratio of apo to holo form
(see next section). The most obvious strategy for resolving these
issues would be to resort to “cofactor engineering,” by genetically
modifying the host so that the cofactor assembly system is present
or that the level of native cofactor assembly is in sufficient supply.
Consider the expression of the clostridial Fe–Fe hydrogenase.
This enzyme depends on an H-cluster that essentially is a di-iron
arrangement with three carbon monoxide, two cyanide ligands,
and one dithiolate bridge (Mulder et al., 2011). The formation
of this cluster is catalyzed by three maturation enzymes; namely
HydE, HydF, and HydG (Posewitz et al., 2004). Since E. coli is
not naturally endowed with the Hyd maturation enzymes, E. coli
invariably produces a non-functional Fe–Fe hydrogenase. This can
be circumvented by simply complementing the expression of Fe–
Fe hydrogenases with the maturation pathway for the H-cluster in
order to form the active Fe–Fe hydrogenase (Posewitz et al., 2004;
Akhtar and Jones, 2008b). Pyrroloquinoline (PQQ) is another
example of a cofactor, which is not naturally synthesized in E.
coli (Matsushita et al., 1997). This cofactor, present in a family
of quinoproteins, has potential uses in biofuel cells, bioremediation, and biosensing (Matsushita et al., 2002). By incorporating the
pqqABCDE gene cluster from Gluconobacter oxydansa, Yang et al.
(2010) were able to successfully demonstrate the activity of a PQQrequiring d-glucose dehydrogenase in E. coli. They noted, however,
that the gene cluster was most likely complemented by the native
tldD gene. A final example is tetrahydrobiopterin, which in itself
is a desirable commodity for the treatment of mild and moderate
forms of phenylketonuria (Perez-Duenas et al., 2004). It can be
synthesized in vivo from GTP via a three-step pathway comprising
GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase, and
sepiapterin reductase (Yamamoto et al., 2003). By augmenting the
pathway with expression of a GTP cyclohydrolase I sourced from
Bacillus subtilis, a 1.5-fold improvement was observed with titers
reaching as high as 4 g biopterin per liter of culture (Yamamoto
et al., 2003).
COFACTOR INSERTION IS KEY TO MAXIMIZING TOTAL AND
SPECIFIC HOLOENZYME ACTIVITY
To maximize the specific activity of a holoenzyme, cofactor synthesis would need to be complemented and/or coupled with
cofactor insertion. For enzymes, which bind to cofactors in a noncovalent fashion, the process of cofactor insertion is somewhat of
an enigma. It is still not known, even for the well-known heme
b cofactor, whether this process is facilitated by dedicated in vivo
components or is a spontaneous process (Thöny-Meyer, 2009).
Though overwhelming in vitro data show that heme b insertion
can be a spontaneous event, recent evidence with in vivo model systems have implicated the involvement of cellular factors that have
yet to be elucidated (Waheed et al., 2010; Correia et al., 2011). For
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Cofactor engineering for metabolic pathways
those enzymes that have covalently attached cofactors, specialized
maturation systems have evolved to catalyze both insertion and
covalent linkage of the cofactor. Induction of the maturation system can increase holoenzyme activity, as in the case of a carboxylic
acid reductase (CAR), which was recently employed for the production of a broad range of chemical commodities (Akhtar et al.,
2013). Venkitasubramanian et al. (2007) had verified that CAR
requires a cofactor known as phosphopantetheine. This cofactor,
during its synthesis, is concomitantly integrated with the enzyme,
via a phosphodiester bond, by a maturation enzyme known as
phosphopantetheinyl transferase. The sole expression of CAR in
E. coli leads to an observable, but exceedingly poor, activity. However, with coexpression of the phosphopantetheinyl transferase Sfp
from Bacillus subtilis, the specific activity of CAR can be enhanced
several-fold to a level that is on par with one that is purified from
the native organism.
In addition to stimulating the specific enzyme activity, increasing the intracellular levels of cofactors is also known to improve the
overall production levels of holoenzymes, suggesting a relationship
between holo/apo-forms and degradation. This is a phenomenon
that has been frequently observed for hemoproteins (Harnastai
et al., 2006; Lu et al., 2010, 2013; Michener et al., 2012). By
elevating heme levels, via supplementation of the media with
δ-aminolevulinic acid, the expression levels of hemoglobin and
cytochrome b 5 can be significantly improved (Gallagher et al.,
1992; Liu et al., 2014). Likewise, increasing Fe–S levels, via overexpression of the isc (Fe–S cluster) operon, also leads to similar
effects (Nakamura et al., 1999; Akhtar and Jones, 2008a). An explanation for the increased holoenzyme levels may be gleaned from
studies of proteins, which utilize divalent metal ions as cofactors
(Wilson et al., 2004; Bushmarina et al., 2006; Palm-Espling et al.,
2012). Evidence from these published reports suggests that cofactors may aid in the folding of the polypeptide chain and, in turn,
accelerate the formation of a functional protein (Goedken et al.,
2000). In the case of ribonuclease HI, metal cofactor integration
was found to impart a greater degree of rigidity on the final native
conformational state of the protein, in addition to improving the
refolding rate of the protein (Wittung-Stafshede, 2002). Further
insights on the importance of cofactors in protein folding can
be gained with the S-adenosylmethionine-containing biotin synthase. This enzyme relies on an intact Fe–S cluster for the addition
of sulfur to dethiobiotin to form the biotin thiophane ring. Reyda
et al. (2008) noticed that the loss of the Fe–S cluster destabilized
the protein, which led to transient unfolding of specific regions, as
well as increased proteolysis. Proteolytic degradation was found to
proceed by an apparent ATP-dependent proteolysis mechanism,
via sequential cleavage of small C-terminal fragments. Interestingly, it was also speculated that since the activity of the protein
is generally well maintained under high-iron conditions, a repair
process, possibly mediated by the Isc and/or Suf (Sulfur mobilization) machinery, may be active under conditions of destabilization
(Reyda et al., 2008).
STIMULATING COFACTOR SYNTHESIS CAN ENHANCE THE
FLUX OF SYNTHETIC PATHWAYS
With regard to the actual impact that cofactor engineering can
have on the metabolic performance of synthetic pathways, two
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Akhtar and Jones
studies are particularly worthy of mention. In the first study relating to the production of the vitamin C precursor, 2-keto-l-gulonic
acid, Gao et al. (2013) utilized two PQQ-dependent dehydrogenases with d-sorbitol as the starting substrate. The authors
noted that induced expression did not improve titers beyond a
certain threshold and hypothesized that PQQ was the limiting
factor. This was proven to be correct since induction of a pathway
for PQQ synthesis resulted in a 20% increase in overall titer. A
later refinement of the work in which the two pathway enzymes
were incorporated as a fusion protein in Ketogulonigenium vulgare also resulted in a similar improvement in titer (Gao et al.,
2014).
The second study relates to a synthetic pathway for hydrogen
production consisting of a pyruvate:ferredoxin oxidoreductase
(PFOR, also known as YdbK in the literature), Fdx and Fe–Fe
hydrogenase (Akhtar and Jones, 2009). The design of this pathway
was based on the observation that pyruvate, rather than NAD(P)H,
was a metabolically superior source of electrons for hydrogen
synthesis (Veit et al., 2008). Initial structural analysis of the PFORbased pathway, in addition to the maturation enzymes, revealed
that each protein component was associated with at least one Fe–S
cluster that essentially provides the route of electron transfer from
pyruvate to the H-cluster of the Fe–Fe hydrogenase (Figure 1B).
Up to a total of 12 Fe–S clusters were found to be required for the
pathway. In E. coli, the formation of Fe–S clusters is undertaken
by the isc operon, though an analogous suf operon is also present
(Fontecave et al., 2005; Roche et al., 2013). Since the isc operon is
controlled by the negative IscR transcriptional regulator, our initial
work using a ∆iscR background strain had shown that the levels
of holo-ferredoxin and the in vitro hydrogenase activity could be
improved two and threefold, respectively, relative to the wild-type
strain (Akhtar and Jones, 2008a). Based on this insight and given
the heavy demand for Fe–S clusters, we reasoned that the ∆iscR
strain may improve the pathway flux toward hydrogen production
by increasing the availability of Fe–S clusters. In accordance with
our prediction, we observed a twofold improvement in hydrogen
yield relative to the wild-type, resulting in an overall yield of 1.5
moles of H2 per mole of glucose. Even more remarkably with addition of TPP, which serves as a cofactor for the PFOR component,
the hydrogen yield was further increased to give a final yield of
1.9, out of a theoretical yield of two moles per mole of glucose
(Akhtar and Jones, 2009). Given also that both the specific and
total in vitro activity of PFOR was improved, this suggests that
the availability of the TPP cofactor may well be another potential limiting cofactor for hydrogen production (Akhtar and Jones,
2009).
Although cofactor biosynthesis and integration under native
control can be limiting and unresponsive to high-expression levels of the apoform, presumably to conserve cellular resources,
cofactor engineering can quite clearly be advantageous for the
assembly of metabolic pathways that depend on the activity of
heterologous holoenzymes. This benefit presumably arises from
the improved holoenzyme activity, via increased structural stability and/or folding rates in conjunction with reduced protein
degradation and/or protein unfolding (explained in the previous section). Interestingly, data from our work on the in vitro
Frontiers in Bioengineering and Biotechnology | Synthetic Biology
Cofactor engineering for metabolic pathways
activity of Fe–S proteins also suggests that, under certain conditions, a steady and constant supply of cofactors may well aid the
restoration of damaged or inactivated enzymes (Akhtar and Jones,
2008a).
CONCLUDING REMARKS
Achieving a balanced production of polypeptide and cofactor
for optimal holoenzyme activity would be an iterative process
involving the (i) modulated induction of genes, (ii) monitoring
of cofactor levels, (iii) evaluation of enzyme activity, and (iv) evaluation of whole-system productivity. Genetic modulation can be
controlled at the transcriptional and translational levels by varying
the strengths of promoter and ribosomal binding sites, while cofactor levels can be monitored and quantified using suitable analytical
methods, e.g., mass spectrometry, high-performance liquid chromatography (HPLC). Combining this with information relating to
specific enzyme activity should allow provide profound insights
into the intracellular cofactor levels required to achieve optimal
holoenzyme activity. Furthermore, if a product reporter system
is available, it may also be possible to screen for optimal cofactor
metabolism, for example using RBS-variation libraries (Zelcbuch
et al., 2013).
Given the importance of cofactor synthesis and integration for
holoenzyme activity, a few key points need to be considered when
assembling pathways involving holoenzymes. Firstly, holoenzyme
activity will only be possible within a host that is metabolically
equipped to synthesize the necessary cofactor, otherwise a complementary pathway for cofactor production would also need to
be implemented. This is particularly relevant for synthetic pathways that employ holoenzymes from diverse origins. Secondly,
to ensure maximal activity of the holoenzyme, the apoenzyme
needs to be sufficiently coupled with the synthesis and insertion of its respective cofactor. An imbalance between the two
will lead to poor enzyme activity, one that would most likely be
inadequate for catalytic purposes. Even native cofactor biosynthesis may not be optimally tuned or responsive to demand from
an over-expressed apoenzyme, as would be required in a biocatalytic system where the objective function has shifted from
biomass to metabolite producer. Thirdly, since cofactor stimulation is known to improve the stability and activity of holoenzymes, cofactor engineering is likely to be a useful strategy for
enhancing the total activity of all holoenzymes in the engineered
pathway, and maximizing chances for high flux toward the product of interest. As it currently stands, the impact of cofactor
engineering on the activity of holoenzymes is still very much a
poorly studied area and certainly warrants more attention given
its potential impact on the success of engineered biocatalytic
systems.
ACKNOWLEDGMENTS
This work that spurred our views, reflected within this perspective, was supported in part by the Academy of Finland
project no. 25369 and European Research Council under the
European Union’s Seventh Framework Programme (FP7/20072013)/European Research Council Grant Agreement 260661
(Patrik R. Jones).
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Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Frontiers in Bioengineering and Biotechnology | Synthetic Biology
Cofactor engineering for metabolic pathways
Received: 12 June 2014; accepted: 12 August 2014; published online: 28 August 2014.
Citation: Akhtar MK and Jones PR (2014) Cofactor engineering for enhancing the flux
of metabolic pathways. Front. Bioeng. Biotechnol. 2:30. doi: 10.3389/fbioe.2014.00030
This article was submitted to Synthetic Biology, a section of the journal Frontiers in
Bioengineering and Biotechnology.
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August 2014 | Volume 2 | Article 30 | 6